Medicinal composition for ophthal use containing acetylated hyaluronic acid

ABSTRACT

The present invention provides an ophthalmic pharmaceutical composition containing acetylated hyaluronic acid and a pharmacologically acceptable carrier. Preferably, the average molecular weight of the acetylated hyaluronic acid is 10,000 to 1,000,000, and the acetyl group substitution number is from 2.0 to 4.0. In a preferred embodiment, this ophthalmic pharmaceutical composition is used in the treatment or prevention of dry eye, and in an even more preferred embodiment it is a dry-eye instillation.

This application claims the priority of Japanese application No.2003-051161 filed on 27^(th) Feb. 2003, which is incorporated byreference.

FIELD OF THE INVENTION

The present invention relates to an ophthalmic pharmaceuticalcomposition that contains acetylated hyaluronic acid and has excellentmoisture retention.

BACKGROUND OF ART

The number of patients or pseudo-patients complaining of dry eye(dryness of the eyes) is expected to continue to climb as our societyages and as people increasingly strain their eyes by watching televisionand using office equipment such as word processors and personalcomputers. The cause of dry eye is a reduction in lacrimal quantity or aqualitative change in the components thereof, and this disorder canresult in scratching or other damage when the surface of the eye becomesdry. Symptoms include eye pain or fatigue, increased blinking, andbloodshot eyes. Further, bacteria may enter through a scratch and causeinfection, and if the scratch is deep enough it can even affect thevision of the person. In addition to eyestrain, causes of dry eyeinclude Sjogren's syndrome, Stevens-Johnson syndrome, burns and injuryto the eye, and side effects of hypotensive drugs, tranquilizers,eyedrops for treating glaucoma, and other such drugs.

Eyedrops are an effective way to treat dry eye, and dry-eye treatmentdrugs whose main component is hyaluronic acid are currently attractingmuch attention and are widely used. Hyaluronic acid is a biologicallyderived macromolecular substance, has extremely high water retention andcharacteristic properties such as a high viscoelasticity, a goodthickening property, and a good thread-forming, ability, and has beenused as a humectant in topical agents for treating various kinds of skinproblems and so forth. In the case of dry eye caused by Sjogren'ssyndrome, in which dryness is seen over the entire body, the applicationof eyedrops containing hyaluronic acid is effective all by itself, butwhen the symptoms are severe, hyaluronic acid-containing eyedrops alonemay not provide a sufficient effect, and it is sometimes necessary forthe treatment to entail tear duct blockage by means of a tear duct plug.Also, hyaluronic acid has a relatively short residence time on thecornea, so the effect of hyaluronic acid eyedrops lasts only about 2 or3 hours, which means that the patient must apply the drops morefrequently (such as 5 to 10 times a day). There is therefore a need foran ophthalmic pharmaceutical composition that would provide along-lasting humectant effect, and therefore require fewer applicationsand improve the QOL (quality of life) of the patient.

Japanese Laid-Open Patent Application S62-68402 discloses a derivativeof hyaluronic acid, namely, a hyaluronic acid that has undergone partialesterification of its carboxyl groups, and suggests that this derivativeis effective as a drug carrier. However, it has yet to be confirmed thatan ophthalmic preparation containing this carboxyl group partiallyesterified hyaluronic acid derivative as a carrier is effective inpharmaceutical compositions used for dry eye, which must provide along-lasting humectant effect.

DISCLOSURE OF THE INVENTION

It is an object of the present invention to provide pharmaceuticalcomposition for dry eye, which remains on the cornea and exhibits ahumectant effect for a longer period.

The inventors were surprised to learn that when an acetylated derivativeof hyaluronic acid is used as an ophthalmic pharmaceutical composition,this derivative stays longer than hyaluronic acid on a soft contactlens, which is a model for the corneal epithelium. Thus, an ophthalmicpharmaceutical composition that contains acetylated hyaluronic acid wasfound to exhibit a humectant effect over a longer period than anophthalmic pharmaceutical composition containing just hyaluronic acid.

Therefore, the present invention provides an ophthalmic pharmaceuticalcomposition containing acetylated hyaluronic acid and apharmacologically acceptable carrier. Preferably, the average molecularweight of the acetylated hyaluronic acid is from 10,000 to 1,000,000,and the acetyl group substitution number is 2.5 to 4.0, even morepreferably the average molecular weight of the acetylated hyaluronicacid is approximately 100,000 and the acetyl group substitution numberis approximately 3.3. In a preferred aspect, this ophthalmicpharmaceutical composition is used for the treatment or prevention ofdry eye, and in an even more preferred aspect, it is a dry-eyeinstillation.

BEST MODE FOR CARRYING OUT THE INVENTION

When applied to the eye, the acetylated hyaluronic acid-containingophthalmic pharmaceutical composition pertaining to the presentinvention exhibits better moisture retention than an ophthalmicpharmaceutical composition containing hyaluronic acid. The cornealepithelium of the eye is composed of about five cell layers, and it someways is similar to the horny layer of the skin, but the cellular surfaceof the topmost layer of the cornea is not at all hydrophilic, and infact is believed to be somewhat hydrophobic. This hydrophobic propertycan also be predicted from the fact that the lacrimal film has athree-layer structure comprising an oil layer (the uppermost layer), awater layer (the middle layer), and a mucous layer (the lowermost layer)that is contact with the corneal epithelium, and mutin, which is amucous component made up primarily of polysaccharide, is present in thislowermost layer. Because its hydroxyl groups have been acetylated,acetylated hyaluronic acid is more highly hydrophobic than hyaluronicacid. Therefore, while not particularly bound to theory, the reason thatthe moisture retention of acetylated hyaluronic acid is better than thatof hyaluronic acid, that is, that acetylated hyaluronic acid remains onthe cornea longer than hyaluronic acid does, is believed to be thatacetylated hyaluronic acid is hydrophobic just like the corneal layer.Also, because it is so highly hydrophilic, there is the danger thathyaluronic acid will absorb moisture from the corneal epithelial tissue.In this regard, acetylated hyaluronic acid is believed to absorb lessmoisture from the corneal epithelial tissue because it is somewhat lesshydrophilic than hyaluronic acid.

The acetylated hyaluronic acid in the present invention is a derivativein which the hydroxyl groups of hyaluronic acid have been acetylated,and more specifically has the following structural formula.

n is a natural number

Acetylated hyaluronic acid can be manufactured by any method known topersons skilled in the art, and examples of such methods are disclosedin Japanese Laid-Open Patent Application H8-53501.

Preferably, the acetylated hyaluronic acid in the present invention isobtained by suspending powdered hyaluronic acid in an acetic anhydridesolvent, and adding concentrated sulfuric acid to bring aboutacetylation. With this method, an acetylated hyaluronic acid with a highdegree of acetylation can be obtained if acetic acid is mixed with theacetic anhydride solvent and the ratio of acetic acid to aceticanhydride is set between 1:4 and 1:1. Also, acetylation will proceedmore moderately and the degree of acetylation can be fine tuned ifacetic acid is mixed with the acetic anhydride solvent and the ratio ofacetic acid to acetic anhydride is set between 2:1 and 4:1. Theconcentrated sulfuric acid is preferably added to the solvent in anamount of 2 to 7 vol %.

There are no particular restrictions on the acetyl group substitutionnumber of the acetylated hyaluronic acid pertaining to the presentinvention, but 2.5 to 4.0 is preferable, 2.8 to 3.8 is even better, and3.0 to 3.8 is better yet. Ideally, the acetyl group substitution numberof the acetylated hyaluronic acid pertaining to the present invention isapproximately 3.3. It is believed that the humectant effect attributableto hyaluronic acid being inherently hydrophilic will be lost if theacetyl group substitution number is too high, but that the humectanteffect attributable to acetylated hyaluronic acid being inherentlyhydrophobic (which is assumed because the cornea is hydrophobic) will belost if the acetyl group substitution number is too low. There are fouralcoholic hydroxyl groups present in the repeating units of hyaluronicacid, as indicated by Chemical Formula 1 above, and “acetyl groupsubstitution number” refers to how many of these have been substitutedwith an acetyl group.

The above-mentioned acetylated hyaluronic acid can be refined intohigh-purity acetylated hyaluronic acid by adding crude acetylatedhyaluronic acid to an acetone aqueous solution, adding and dissolvingsodium lactate, and adding a high concentration of acetone. Theabove-mentioned sodium lactate is preferably added in an amount of 1 to3 wt % with respect to the acetone aqueous solution.

“Hyaluronic acid” as used in the present invention means hyaluronic acidand hyaluronates, and types of various molecular weight can be used.There are no particular restrictions on the average molecular weight ofthe acetylated hyaluronic acid pertaining to the present invention, butit is preferably 1,000 to 1,000,000, and even more preferably 10,000 to1,000,000. The ideal average molecular weight is approximately 100,000.

The amount of acetylated hyaluronic acid contained in the ophthalmicpharmaceutical composition of the present invention is 0.01 to 10weight/volume (weight)%, and preferably 0.05 to 5 weight/volume(weight)%, and even more preferably 0.1 to 1 weigh/volume (weight)%,based on the total volume (or total weight) of the pharmaceuticalcomposition. The acetylated hyaluronic acid concentration refers to theweigh/volume (w/v)% in the case of a liquid eye wash or eyedrops, and toweigh/weight (w/w)% in the case of a solid eye ointment. While it willdepend on the hyaluronic acid and its molecular weight, an aqueoussolution in which the concentration is over 3% usually cannot be usedfor eyedrops because its viscosity will be too high, but preparing anaqueous solution of acetylated hyaluronic acid with a concentration ofabout 10% is quite possible.

The pH of the ophthalmic pharmaceutical composition of the presentinvention is preferably close to neutral, and is usually from 6.5 to7.5. The osmotic pressure ratio is preferably adjusted to about 0.5 to4.0, with a range of 1.0 to 1.5 being even better. Any means commonlyemployed for ophthalmic pharmaceutical compositions can be used toadjust the pH and osmotic pressure.

The ophthalmic pharmaceutical composition pertaining to the presentinvention is effective against symptoms of eye dryness, and particularlyin the treatment and prevention of dry eye. Dry eye can be caused by avariety of factors, such as eyestrain, Sjogren's syndrome,Stevens-Johnson syndrome, burns and injury to the eye, and side effectsof hypotensive drugs, tranquilizers, eyedrops for treating glaucoma, andother such drugs. The ophthalmic pharmaceutical composition of thepresent invention is formulated as eyedrops, an eye wash, an eyeointment, or the like.

Depending on its formulation, the ophthalmic pharmaceutical compositionof the present invention can also be used together with apharmacologically acceptable carrier. The carrier for eyedrops and eyewashes can be any type ordinarily used for such purposes, and purifiedwater is favorable.

The ophthalmic pharmaceutical composition of the present invention mayoptionally be compounded with various components other than acetylatedhyaluronic acid, such as sugars, electrolytes, amino acids, vitamins,lipids, medicinal additives, and medicines. Examples of these componentsinclude sugars such as glucose, maltose, etc., oligosaccharides,mannitol, and sugar alcohols such as sorbitol; electrolytes such assodium chloride, sodium hydrogenphosphate, potassium chloride, magnesiumsulfate, and calcium chloride; amino acids such as glycine and alanine;vitamins such as thiamin hydrochloride, sodium riboflavin phosphate,pyridoxine hydrochloride, nicotinic acid amide, folic acid, biotin,vitamin A, L-ascorbic acid, and α-glycosyl ascorbic acid; andderivatives of these. These may be compounded in suitable combinationsas needed.

As long as the humectant effect of the present invention is notcompromised, it is also possible to add any additives that are used inordinary ophthalmic preparations, examples of which includepreservatives such as methyl parahydroxybenzoate, sodium dehydroacetate,and benzalkonium chloride; stabilizers such as sodium edetate and sodiumhydrogensulfite; buffers such as borax, boric acid, and sodiumhydrogencarbonate; thickeners such as methyl cellulose, carboxymethylcellulose, chondroitin sulfuric acid, polyvinyl alcohol, and pullulan;and dissolution improvers such as Polysorbate 80.

If the ophthalmic pharmaceutical composition of the present invention isan ointment, any commonly used and pharmacologically acceptable ointmentcarrier can be used, specific examples of which include ophthalmic whitevaseline and plastibase. Liquid paraffin or the like may also be used asan additive. The ophthalmic pharmaceutical composition of the presentinvention may additionally contain as needed methylprednisolone andother such steroidal hormones, tetracycline and other suchantiphlogistics, penicillin G and other such antibiotics, cyclosporinand other such immunosuppressants, and pharmaceutical products such asimmunomodulators, analgesics, autoserum, and hyaluronic acid.

The dosage and method of administration of the ophthalmic pharmaceuticalcomposition of the present invention can be suitably adjusted accordingto the symptoms of the patient. In the case of an instillation, usuallyabout one to four drops (assuming one drop to be about 0.05 mL) areadministered each time, either once or several times daily (such as oneto five times, and preferably one to three times). In the case of an eyewash, the patient should use a special eye wash container, a washbottle, or the like, and wash the eyes from one to several times a day(such as one to five times, and preferably one to three times). In thecase of an eye ointment, the inside of the conjunctival sac should becoated with a suitable quantity about one to three times a day.

The use of acetylated hyaluronic acid makes it possible to provide anophthalmic pharmaceutical composition for dry eye, which remains on thecornea and exhibits a humectant effect over a longer period than whenhyaluronic acid is contained.

Examples of the present invention will now be given, but do not limitthe scope of the present invention.

EXAMPLES

In their research for the present invention, the inventors examined theability of acetylated hyaluronic acid (hereinafter referred to as“AcHA”) to suppress moisture evaporation, using a soft contact lens(SCL) as a substitute for a human or animal cornea, in an attempt toapply AcHA to an ophthalmic pharmaceutical composition used for thetreatment of dry eye.

Experimental Materials and Methodology

The SCLs used as a substitute cornea were Focus Dailies (disposable,13.8 mm diameter) from Ciba Vision (Tokyo).

The SCLs used in the experiment were made from Nelfilcon A, which is ahydrogel based on a therapeutic material (polyvinyl alcohol, PVA) thathas good biocompatibility and is used for surgical sutures andartificial blood vessels. This SCL retains a large amount of water (69%)and has satisfactory oxygen permeability. Accordingly, this SCL has goodbiocompatibility and good affinity with the cornea, making it suitableas a substitute cornea in physical property research such as this (suchas measuring the amount of moisture evaporation).

An isotonic phosphate buffer saline (PBS) (prepared from Dulbecco's PBS(−) powder “Nissui” made by Nissui Pharmaceutical (Tokyo)) was used as acontrol instillation, while Hyalein 0.1 (containing 0.1 w/v % sodiumhyaluronate; average molecular weight of hyaluronic acid: 600,000 to1,200,000; made by Santen Pharmaceutical (Osaka)) was used as ahyaluronic acid (HA) instillation. The AcHA instillation was prepared bydissolving AcHA (estimated average molecular weight of approximately100,000; acetylation substitution number of approximately 3.5) in PBS sothat the final concentration would be 0.1 w/v %.

The AcHA was prepared as follows.

Twenty milliliter of commercially available special grade acetic acidand 80 mL of acetic anhydride were put in an Erlenmeyer flask with avolume of 300 mL, to which 6 g of a fine powder of hyaluronic acid(average molecular weight of approximately 1200 kd, made by Shiseido)was added a little at a time while stirring. Four milliliter ofconcentration sulfuric acid was then added slowly, and the system wasstirred for 1 hour at room temperature to bring about an acetylationreaction. The reaction solution was in the form of a viscous whiteliquid.

Two liter of purified water was put into a 3 L glass beaker, and theabove-mentioned reaction solution was slowly added in the form of a finestream while stirring. The precipitate of acetylated hyaluronic acidthus produced was collected and washed twice with 2 L of purified water.This precipitate was then transferred to a 1 L glass beaker, 250 mL ofan aqueous 80% (v/v) acetone solution and 9 g of a 50% sodium lactateaqueous solution were added, and the precipitate was completelydissolved under stirring. Four hundred milliliter of acetone was thenadded slowly, and a gel of acetylated hyaluronic acid wasreprecipitated. This precipitate was collected, after which it waswashed twice for 10 minutes each time in a homogenizer at a speed of10,000 rpm and using 100 mL of ethanol. The precipitate was thencollected by reduced pressure filtration, after which it was dried underreduced pressure, which gave a white powder of acetylated hyaluronicacid.

Measurement Method

An AG64 balance made by Mettler Toledo was used to weigh the SCL, andweight data were sent to and stored in a personal computer.Nonparametric testing, which is not affected by the distribution ofdata, was employed for the statistical analysis of the data.

The experiments (the application of the eyedrops on SCLs, leaving themin the eyes, and measuring the weight of SCLs) were conducted in athermohygrostatic room at a temperature of 25° C. and a humidity of 50%.Each SCL packaged in a isotonic phosphate buffer was taken out withdental forceps and placed convex-side up on the pre-weighed lid (30 mmdiameter) of a plastic petri dish, the weight of the petri dishincluding the SCL was measured, and 20 μL of sample instillation wasimmediately dropped onto the center of the SCL. After 20 minutes hadelapsed, the weight of the petri dish including the SCL was measured,and another 20 μL of sample instillation was immediately dropped ontothe center of the SCL. This cycle of measurement and sample dropping wasrepeated a total of four times at 20 minute intervals. After the finalsample dropping, the SCL was weighed every 20 minutes until 120 minutesafter the start of the experiment.

Experiment Results

As shown in Table 1 below, the SCLs in the AcHA instillation group at120 minutes after the start of the experiment had significantly lowermoisture evaporation amounts than the control instillation group or theHA instillation group. TABLE 1 Relative wet weight (%) of soft contactlens after several instillations Time Control HA AcHA (min) instillationinstillation instillation 80 130.4 145.2*^(a) 149.2*^(b) 100 105.1118.1*^(a) 123.9*^(b) 120 82.13  86.03  99.8*^(b,)*^(c)*^(a)with respect to the control instillation group; p < 0.05)*^(b)with respect to the control instillation group; p < 0.05)*^(c)with respect to the HA instillation group; p < 0.05)

The numerical values are the median value for each group, and the numberof observations was 5 for the control instillation group and HAinstillation group, and 4 for the AcHA instillation group. TheMann-Whitney method (a nonparametric test) was used for a significantdifferent test.

CONCLUSION

The evaporation of moisture from an SCL was significantly suppressedwith a 0.1% AcHA instillation. It is clear from this that the AcHAinstillation has the potential for application as an instillation in thetreatment of dry eye. AcHA instillation composition acetylatedhyaluronic acid 0.1 g benzalkonium chloride 0.002 g isotonic phosphatebuffer q.s. total 100 mL

With the above composition, AcHA can be contained in an amount of about0.1 to 10 g, and since AcHA has a high molecular weight (approximately100,000), even at a high concentration it will be unlikely to have aneffect on the osmotic pressure ratio of the instillation.

1. An ophthalmic pharmaceutical composition comprising acetylatedhyaluronic acid and a pharmacologically acceptable carrier.
 2. Theophthalmic pharmaceutical composition according to claim 1, wherein theaverage molecular weight of the acetylated hyaluronic acid is from10,000 to 1,000,000, and the acetyl group substitution number is 2.0 to4.0
 3. The ophthalmic pharmaceutical composition according to claim 1,which is used for the treatment of dry eye.
 4. The ophthalmicpharmaceutical composition according to claim 3, which is a dry-eyeinstillation.
 5. The ophthalmic pharmaceutical composition according toclaim 2, which is used for the treatment of dry eye.